title: Reactive thioglucoside substrates for β-glucosidase.
abstract: A new, very efficient, class of thioglycoside substrates has been found for β-glucosidase. While thioglycosides are usually resistant to hydrolysis, even in the presence of acids or most glycohydrolases, the β-D-glucopyranosides of 2-mercaptobenzimidazole (GlcSBiz) and 2-mercaptobenzoxazole (GlcSBox) have been found to be excellent substrates for β-glucosidase from both sweet almond (a family 1 glycohydrolase) and Aspergillus niger (a family 3 glycohydrolase), reacting nearly as well as p-nitrophenyl β-D-glucoside. The enzyme-catalyzed hydrolysis of GlcSBiz proceeds with retention of configuration. As with the (1000-fold slower) hydrolysis of phenyl thioglucosides catalyzed by the almond enzyme, the pL (pH/pD)-independent kcat/KM does not show a detectable solvent deuterium kinetic isotope effect (SKIE), but unlike the hydrolysis of phenyl thioglucosides, a modest SKIE is seen on kcat [(D2O)kcat=1.28 (±0.06)] at the pL optimum (5.5≤pL≤6.6). A solvent isotope effect is also seen on the KM for the N-methyl analog of GlcSBiz. These results suggest that the mechanism for the hydrolysis of the β-thioglucoside of 2-mercaptobenzimidazole and of 2-mercaptobenzoxazole involves remote site protonation (at the ring nitrogen) followed by cleavage of the thioglucosidic bond resulting in the thione product.
authors.affiliation: Department of Chemistry, Tulane University, New Orleans, LA 70118, United States.
authors.forename: Elizabeth Alverson-Banks
authors.initials: EA
authors.lastname: Avegno
authors.forename: Scott J
authors.initials: SJ
authors.lastname: Hasty
authors.forename: Archana R
authors.initials: AR
authors.lastname: Parameswar
authors.forename: Gary S
authors.initials: GS
authors.lastname: Howarth
authors.forename: Alexei V
authors.initials: AV
authors.lastname: Demchenko
authors.forename: Larry D
authors.initials: LD
authors.lastname: Byers
mesh_terms: D001665:Binding Sites; D004789:Enzyme Activation; D004795:Enzyme Stability; D011485:Protein Binding; D013379:Substrate Specificity; D013863:Thioglucosides / Q000737:chemistry*; D001617:beta-Glucosidase / Q000737:chemistry*
chemical_list: D013863:Thioglucosides; D001617:beta-Glucosidase
keywords: 2-Mercaptobenzimidazoyl β-thioglucopyranoside; Retention; Solvent kinetic isotope effect; β-Glucosidase